System for rapid nucleic acid amplification and detection

ABSTRACT

A system is provided for carrying out rapid nucleic acid amplification or other biological reactions requiring thermal cycling. The system of this invention incorporates at least two heating blocks, each having a groove for receiving a reaction vessel such that only a portion of the outer surface of the walls of the vessel are in direct contact with the heating block. The remaining portion of the outer surface of the walls of the reaction vessel remains exposed to ambient conditions. The reaction vessel comes into contact with only one heating block at a time either by movement of the vessel between the heating blocks or by movement of the heating blocks in relation to the vessel. The system of this invention provides rapid temperature cycling without the need for extended ramping times generally associated with single block designs, which include a single temperature block that is forced to heat and cool. Heating and cooling of the reaction using the system of the present invention is accomplished by the reaction vessel and heating blocks coming into thermal contact and reaching thermal equilibrium. The entire vessel need not be surrounded by the heating block, with at least one side of the vessel partially open to ambient conditions. As a result of the configuration of this system it is readily combined with detection systems, for example, fluorescence detection systems.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation-in-part of co-pending U.S. national phase application Ser. No. 11/578,440 entered nationally on Oct. 13, 2006, and filed internationally on Apr. 15, 2005 as PCT International Application No. PCT/CA2005/000576, which claim the benefit of U.S. provisional application Ser. No. 60/563,061 filed on Apr. 16, 2004, and both of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a temperature cycling apparatus useful for performing nucleic acid amplification and detection. More specifically, the present invention relates to a thermal cycling apparatus for rapidly cycling the temperature of a sample through a predetermined temperature cycle.

BACKGROUND OF THE INVENTION

In numerous areas of industry, technology, and research there is a need to reliably and reproducibly subject relatively small samples to thermal cycling. The need to subject a sample to repeated temperature cycles is particularly acute in biotechnology applications. In the biotechnology field, it is often desirable to repeatedly heat and cool small samples of materials over a short period of time. One such biological process that is regularly carried out is cyclical DNA amplification.

Cyclic DNA amplification, using a thermostable DNA polymerase, allows automated amplification of primer specific DNA, widely known as PCR, or the polymerase chain reaction. It is well accepted that automation of this process requires controlled and precise thermal cycling of reaction mixtures.

PCR is a technique involving multiple cycles that results in the geometric amplification of certain polynucleotide sequence each time a cycle is completed. The technique of PCR is well known to the person of average skill in the art of molecular biology.

Commercial programmable metal heat blocks have been used in the past to affect the temperature cycling of samples in microfuge tubes through the desired temperature versus time profile. Peltier heating and cooling are usually utilized in changing temperatures at a rate of approximately 0.5-4 degrees Celsius per second. However, the inability to quickly and accurately adjust the temperature of the heat blocks through a large temperature range over a short time period, has rendered the use of heat block type devices undesirable as a temperature control system when carrying out the polymerase chain reaction in a rapid fashion.

A wide variety of instrumentation has been developed for carrying out nucleic acid amplifications. Important design goals fundamental to PCR instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-PCR processing steps, high speed cycling, minimization of sample volumes, real-time measurement of amplification products, minimization of cross-contamination, or sample carryover, and the like.

A prior art system is represented by a temperature cycler in which multiple temperature controlled blocks with vertical reaction vessel wells are maintained at different desired temperatures (U.S. Pat. Nos. 5,525,300, 5,779,981 and 6,054,263). A robotic arm is utilized to move reaction mixtures from block to block. The reaction vessels are lifted vertically from out of the heat block, transported to another heating block, and placed vertically down into said heating block. However, this system requires precision movement, pressurized thermal contact and expensive microprocessor controlled robotics. This robotic movement system also impedes a real-time fluorescent detection system of the nucleic acid amplification product during and after temperature cycling has completed.

Rapid cycling has been described before (e.g. U.S. Pat. No. 6,174,670 and U.S. Pat. No. 5,455,175). According to this prior art, rapid cycling techniques are made possible by the rapid temperature response and temperature homogeneity of samples in high surface area-to-volume sample containers such as capillary tubes. For further information, see also: C. T. Wittwer, G. B. Reed, and K. M Ririe, Rapid cycle DNA amplification, in K. B. Mullis, F. Ferre, and R. A. Gibbs. The polymerase chain reaction, Birkhauser, Boston, 174-181, (1994). According to this prior art, improved temperature homogeneity allows the time and temperature requirements of PCR to be better defined and understood, while improved temperature homogeneity also increases the precision of any analytical technique used to monitor PCR during amplification.

The design of instruments that permit PCR to be carried out in closed reaction chambers and monitored in real-time is highly desirable. Closed reaction chambers are desirable for preventing cross-contamination, e.g. Higuchi et al, Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al, Proc. Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives would severely reduce the value of the PCR-based procedure. Real-time monitoring allows the coupling of amplification and detection, thus decreasing contamination risks and labour time. As well, real-time monitoring of a PCR permits far more accurate measurement of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the PCR. Real-time monitoring also permits the efficiency of the PCR to be evaluated, which can indicate whether PCR inhibitors are present in a sample.

Holland et al (cited above) and others have proposed fluorescence-based approaches to provide real-time measurements of amplification products during a PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present, others have employed probes containing fluorescent-quencher pairs (the so-called “Taq-Man™” approach) that are cleaved during amplification to release a fluorescent product whose concentration is proportional to the amount of double stranded DNA present. Other fluorescent probe technologies have also been used in real-time PCR, including Fluorescent Resonance Energy Transfer (FRET) probes (U.S. Pat. Nos. 6,174,670 and 6,569,627), linear probes in which one probe stimulates and adjacent probes to fluoresce, and molecular beacons in which a hairpin loop is formed within the probe to quench the florescence when the probe is not hybridized to the target nucleic acid.

The accepted state of the art is that nucleic acid reactions, such as PCR, require a uniform temperature in order to be successful. This was expressed in 1998 (by Neumaier et al. and Wagener et al. Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. Clinical Chemistry. 44(1): 12-26) as follows:

-   -   “Uniform temperature transition is an important aspect of         successful amplification”: and     -   “The homogeneity of heat conduction in the reaction block is of         crucial importance. The heat performance of the cycler and the         uniformity of heat conduction in the heating block must be         controlled regularly to avoid false negative results”.

Consequently, manufacturers of PCR machines have engineered their instruments to generate uniform thermal gradients. For example, in 1992, Stratagene introduced the RoboCycler temperature cycler, a unique four-block instrument that claimed to achieve unparalleled temperature uniformity (Renzi, P., et al. (1992) Strategies 5: 41-42). More recently, Corbett Research developed the Rotor-Gene instrument that heats and cools PCR reaction tubes via air jets. The rotor containing the reaction tubes spins at very high speeds, and the stated intention is to increase temperature uniformity. Additionally, there are products such as the DRIFTCON system (Appropriate Technical Resources, Inc.) that enable researchers to test the temperature uniformity of the thermal block in their PCR machines.

SUMMARY OF THE INVENTION

The present invention provides an apparatus for thermal cycling of a reaction sample between two or more temperatures in which heat is applied to a reaction vessel, unequally to the vessel wall. It has been found that unequal application of heat from heating means such as a heating block, to the reaction vessel, permits a more rapid cycling of the reaction vessel and its contents between different reaction temperatures, while still permitting reactions such as PCR to effectively occur.

The prototype device of the present invention was able to successfully perform PCR reactions using an apparatus where the thermal gradient across the reaction tube was highly non-uniform.

Specifically, the prototype apparatus consisted of fixed temperature heat blocks, with an engraved groove that fit one-half of a standard PCR reaction tube. During a temperature cycle, only one side of the PCR reaction tube was in contact with the heat source, while the opposite side was exposed to ambient air temperature. This unequal heating resulted in a large thermal gradient across the reaction tube. Contrary to conventional thinking the PCR reaction was successful, and no non-specific amplicons were seen in post-PCR gel electrophoresis. In fact, gel electrophoresis analysis showed that the amplicon generated by the prototype apparatus using a highly non-uniform thermal gradient was the same as the amplicon generated with the GeneAmp 2700 machine (Applied Biosystems), using a thermal block with a highly uniform temperature gradient.

With the new knowledge that nucleic acid amplification reactions can be successfully performed when the, reaction vessel is subject to a non-uniform temperature gradient, it is possible to design set-ups such as the prototype device, where a reaction tube is moved in a single degree of freedom of motion between two heat blocks, set at fixed temperatures. This makes it possible to do away with the robotics and electronics of the Stratagene RoboCycler, where the tube must be moved in three dimensions so that it can fully (and uniformly) enclosed in the temperature block.

Another advantage is that an entire side of the tube may be conveniently exposed for real-time detection by an optical detection system. This avoids the need for complicated optics that monitor the progress of a real-time PCR reaction by directing excitation light down through the top of the reaction tube, and then collecting emitted light that is reflected back up through the top of the tube, (for example, this is the system used in some of the real-time PCR machines from Applied Biosystems). For example, it is contemplated that a possible design would be to embed the reaction tube partially in a heat block and perform PCR by ramping the temperature of the heat block. At the same, real-time optical detection could be performed by detecting emitted fluorescence from the exposed portion of the reaction tube.

According to one aspect, it is an object of the present invention to provide a thermal cycling apparatus for quickly and accurately varying the temperature of biological samples according to a predetermined temperature versus time profile.

According to another aspect, it is also an object of the present invention to provide a thermal cycling apparatus that can effectively subject samples to a large temperature gradient over a very short period of time.

According to another aspect, it is a further object of the present invention to provide an apparatus that can subject a biological sample to rapid thermal cycling by rapidly alternating the sample between two heating blocks, which may be in opposing relation to one another.

According to another aspect, it is another object of the present invention to provide a thermal cycling apparatus that will heat samples located in a fluid chamber placed in an engraved groove in the side of the heating blocks.

According to another aspect, the two heating blocks are positioned in ambient air, and a portion of the reaction vessel while in the heating block is exposed to air without compromising a thermal gradient within the reaction vessel.

According to another aspect, it is also an object of the invention to provide a real-time nucleic amplification product detection mechanism between the two heating blocks by means of fluorescence monitoring of the product labeled with a fluorescent dye.

Thus according to the present invention there is provided a thermal cycling process for polymerase chain reaction (PCR) wherein a reaction vessel (RV) having a vessel wall—containing a reaction mixture comprising a target nucleic acid (NA) and reagents selected to achieve amplification of said target NA by means of the PCR—is thermally cycled between two predetermined temperatures, the improvement characterized by subjecting the RV to a temperature gradient during said thermal cycling process, and preferably a large temperature gradient which is maintained during all or substantially all of the duration of the cycling step.

In the above process, the heat of each of the two predetermined temperatures to obtain the temperature gradient is applied unequally to the wall of the RV.

Preferably, the heat is applied unequally to the vessel wall by contacting only a portion of the wall with a heating, element while exposing the remainder of the wall to ambient air.

In the process rapid thermal cycling is provided wherein a temperature spectrum of the NA and reagents is maintained within the RV during the thermal cycling process.

Further, preferably annealing and extension of NA occur in a single step during the PCR process.

A PCR device, according to the present invention, for thermal cycling of a reaction mixture contained in a reaction vessel (RV) having an outer surface, comprising at least two heating sources, characterized by each of the at least two heating sources having a different predetermined temperature, for successively heating only part of the outer surface of the RV.

In a narrower aspect in the PCR device the two heating sources are spaced apart heating blocks and each having a groove contoured to receive and provide thermal contact with only a portion of the outer surface of the RV; and the means for causing the RV to be in successive physical thermal contact with each the two heating blocks for predetermined times and for a number of successive contacts sufficient to promote the PCR in the reaction mixture contained in the RV.

The PCR device further comprises a means for maintaining the heating sources at different predetermined temperatures and a means for causing the RV to be in successive physical contact with each of the heating sources for predetermined times and for a number of successive contacts sufficient to promote PCR in the reaction mixture contained in the RV, the heating sources being configured for successively contacting only part of the outer wall of the RV while the remainder of the surface is exposed to ambient air.

In the PCR device the two heating sources comprise spaced apart heating blocks each having a groove contoured to receive and provide thermal contact with only a portion of the outer wall of the RV.

The PCR device further comprises, a controlling means for operating the device so as to achieve a time versus temperature profile which subjects the RV to a large temperature gradient during the thermal cycling process.

In the PCR device the controlling means controls the device to provide annealing and extension of the NA within the RV in a single step during the PCR process by maintaining essentially no holding time at the temperature required for the step.

In the PCR device the two heating blocks are in spaced apart opposing relation to each other and the grooves oppose each other.

The PCR device further comprises a holder for retaining the RV such that when engaged within the groove, the drive means sequentially causes relative movement between the RV and the blocks for successive contact with the opposing grooves.

In the PCR device the grooves are dimensioned to contact between about 40% and 50% of the wall of the RV.

The PCR device further comprises means for real-time florescence-based measurement of a preselected nucleic acid (NA), if present in the reaction mixture during operation of the PCR device.

The apparatus of the present invention includes a controlling means for operating the apparatus through the desired time versus temperature profile. The present invention is particularly well suited for carrying out automated and rapid polymerase chain reactions.

The controller of the present invention allows the chamber, and subsequently the samples located in the sample compartment therein, to pass through a predetermined temperature cycle corresponding to the denaturation and annealing/extension steps in the polymerase chain reaction. In use, the apparatus of the present invention allows rapid optimization of denaturation, and annealing/extension steps in terms of time and temperature, and shortened or minimal time periods (ramp times) between the temperatures at each step.

The present invention particularly decreases the total time required for completion of polymerase chain reaction cycling over prior art thermal cycling devices by having high reaction vessel liquid ramp time (>2° C./sec), by decreasing the number of steps per cycle from three to two (the annealing and extension steps are done at one temperature), and by decreasing the amount of time for each step of each cycle without compromising amplification efficiency.

The invention relates to a system for carrying out rapid nucleic acid amplification using PCR, which can be coupled with real-time fluorescence-based measurements of nucleic acid amplification products. In one preferred embodiment of the invention, an excitation beam is focused into a reaction mixture containing a nucleic acid intercalating dye or hybridization probe which fluoresces proportionally to the amount of nucleic acid amplification product, it is understood that the proportionality of the fluorescent intensities is for a constant set of parameters such as temperature, pH, salt concentration, and the like, that independently influence the fluorescent emissions of organic dyes.

Preferably, the excitation beam is focused into the reaction mixture through a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture.

In one embodiment, the reaction chamber is a reaction vessel with a closed end, referred to herein as the bottom of the vessel, and an open end, referred to herein as the top of the vessel, which can be closed with a cap such that a leak-proof seal is formed. In other words, once a reaction mixture is placed in the vessel and the cap is attached a closed reaction chamber is formed. In this embodiment, (1) the reaction mixture fills a portion of the reaction vessel, generally at the bottom of the vessel, such that a void is left between the cap of the vessel and a top surface of the reaction mixture, and (2) the reaction is completed in the apparatus described above and (3) the lens, without contacting the side of the vessel, allows the excitation beam through the vessel wall into the reaction mixture through its side surface and (4) the resulting fluorescence generated by the fluorescent indicator is collected by a photosensor, such as a photodiode, at various time points.

As discussed more fully below, an excitation beam generated by a single light source, e.g. a Light Emitting Diode (LED), is placed on one side of the vessel. The wavelength of the excitation beam is restricted by appropriately selecting a colored type of LED as well as using a color filter by which LED light travels through. Likewise, a different color filter located on the opposite side of the vessel restricts the resultant emission fluorescence wavelength. A photodiode sensor collects the filtered emission light and the amplitude of the emission is examined and compared by an analysis system.

Preferably, the system is employed with the PCR amplification of nucleic acids.

The system of the invention permits accurate real-time monitoring of nucleic amplification reactions by providing apparatus and fluorescent reagents for generating a stable fluorescent signal proportional to the amount of amplification product and independent of variations in the volume of reaction mixture. The availability of data showing the progress of amplification reactions leads to rapid assessment of the efficiency of the amplification reactions, and opens the possibility of reduced reagent usage and feedback reaction control.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood by reference to the appended figures of which:

FIG. 1: is a perspective view of a portion of a thermal alternator device according to one embodiment of the invention.

FIG. 2A is a perspective view of a thermal alternator according to the first embodiment of the invention.

FIG. 2B is a perspective view of a second embodiment of the invention.

FIG. 3: is a perspective view of the fluorescence detection apparatus according to one embodiment of the present invention.

FIG. 4: is a top plan view of a reaction vessel holder for carrying out a method according to one embodiment of the present invention;

FIG. 5: is an isometric view of the reaction vessel holder shown in FIG. 4;

FIG. 6: is a block diagram of the control and processing electronics of the present invention;

FIG. 7: is a general flow-chart illustrating how the device of the present invention is operated; and

FIGS. 8A & 8B: are flow-charts summarizing the control software of the present device.

DETAILED DESCRIPTION

In describing and claiming the present invention, the following definitions apply:

“Nucleic acid,” “DNA,” and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.

“Amplification” of DNA denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixture of DNA sequences.

An “amplicon” is a product of the amplification of a target genetic sequence.

A “PCR reaction mixture” denotes a mixture adaptable for simultaneously amplifying multiple genetic targets under suitable conditions for PCR.

A “genetic target” denotes a genetic sequence capable of amplification by polymerase chain reaction (PCR). A genetic target in accordance with the present invention includes any DNA sequence, including bacterial, viral, fungal, human, plant, and animal DNA, for example.

“Continuous monitoring” and similar terms refer to monitoring multiple times during a cycle of PCR, preferably during temperature transitions, and more preferably obtaining at least one data point in each temperature transition.

“Fluorescence detection” and similar terms refer to labeling nucleic acids with a fluorescence indicator. The fluorescence indicator can be a nucleic acid intercalating dye such as Ethidium Bromide, Thiazole orange, Pico™ Green or SyBr™ Green. As well, labeled hybridization probes using FRET, Taq-Man™ or other chemistries such as molecular beacons can also be used as fluorescence detection tools.

“Effective amount” means an amount sufficient to produce a selected effect. For example, an effective amount of PCR primers is an amount sufficient to amplify a segment of nucleic acid by PCR provided that a DNA polymerase, buffer, template, and other conditions, including temperature conditions, known in the art to be necessary for practicing PCR are also provided.

“Probe”, refers to a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid, which, in the context of the present invention, is an amplicon, under standard conditions that promote hybridization. This allows detection of the amplicon. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the amplicon sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target amplicon). A probe's “target” generally refers to a sequence within (i.e., a subset of) an amplified nucleic acid sequence which hybridizes specifically to at least a portion of a probe oligomer using standard hydrogen bonding (i.e., base pairing). A probe may comprise target-specific sequences and other sequences that contribute to three-dimensional conformation of the probe (e.g., as described in Lizardi et al., U.S. Pat. Nos. 5,118,801 and 5,312,728).

By “sufficiently complementary” is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another base sequence by hydrogen bonding between a series of complementary bases. By definition, this allows stable hybridization of a probe oligomer to a target sequence in the amplicon even though it is not completely complementary to the probe's target-specific sequence. Complementary base sequences may be complementary at each position in the base sequence of an oligomer using standard base pairing or may contain one or more residues that are not complementary using standard hydrogen bonding (including a basic “nucleotides”), but in which the entire complementary base sequence is capable of specifically hybridizing with another base sequence in appropriate hybridization conditions. Contiguous bases are preferably at least about 80%, more preferably at least about 90%, and most preferably greater than 95% complementary to a sequence to which an oligomer is intended to specifically hybridize. To those skilled in the art, appropriate hybridization conditions are well known, can be predicted based on base composition, or can be determined empirically by using routine testing (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at § 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57 particularly at §9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

The terms “label” and “detectable label” refer to a molecular moiety or compound that can be detected or can lead to a detectable response. A label is joined, directly or indirectly, to a nucleic acid probe. Direct labeling can occur through bonds or interactions that link the label to the probe, including covalent bonds or non-covalent interactions (e.g., hydrogen bonding, hydrophobic and ionic interactions) or through formation of chelates or co-ordination complexes. Indirect labeling can occur through use of a bridging moiety or “linker,” such as an antibody or additional oligonucleotide(s), which is either directly or indirectly labeled, and which can amplify a detectable signal. A label can be any known detectable moiety, such as, for example, a radionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactive group, chromophore, such as a dye or particle that imparts a detectable color (e.g., latex or metal particles), luminescent compound (e.g., bioluminescent, phosphorescent or chemiluminescent labels) and fluorescent compound.

PCR techniques applicable to the present invention include inter alia those described in “PCR Primer. A Laboratory Manual”, Dieffenback, C. W. and Dveksler, G. S., eds., Cold Spring Harbor Laboratory Press (1995); “Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia”, Saiki R K, Scharf S, Faloona F, Mullis K B, Horn G T, Erlich H A, Amheim N, Science (1985) December 20; 230(4732): 1350-4.

The PCR of the present invention is performed using a modified 2-step cycling profile as compared to standard PCR, namely successive cycles of denaturation of double stranded target nucleic acid and annealing/extension of the primers to produce a large number of copies of segments of the target DNA. Each cycle is a thermal cycle in which the reaction temperature is raised to denature the double stranded DNA and lowered to allow annealing and extension.

In one embodiment of the present invention, the PCR makes use of successive two-step cycles in which the temperature is raised to a first temperature for denaturation of the double stranded DNA and lowered to a second temperature to allow annealing and extension of the primers.

Following amplification of a nucleic acid using the system described herein, the amplicons may be detected using any method known in the art.

Preferably, the label on a labeled probe is detectable in a homogeneous assay system, i.e., where, in a mixture, bound labeled probe exhibits a detectable change, such as stability or differential degradation, compared to unbound labeled probe, without physically removing hybridized from non-hybridized forms of the label or labeled probe. A “homogeneous detectable label” refers to a label whose presence can be detected in a homogeneous fashion, for example, as previously described in detail in Arnold et al., U.S. Pat. No. 5,283,174; Woodhead et al., U.S. Pat. No. 5,656,207; and Nelson et al., U.S. Pat. No. 5,658,737. Examples of labels that can be used in a homogenous hybridization assay include, but are not limited to, chemiluminescent compounds (e.g., see U.S. Pat. Nos. 5,656,207, 5,658,737 and 5,639,604), such as acridinium ester (“AE”) compounds, including standard AE or derivatives thereof. Synthesis and methods of attaching labels to nucleic acids and detecting labels are well known in the art (e.g., see Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, 4,581,333 and European Patent Application No. 0 747 706).

In accordance with another embodiment of the present invention, where the amplicons are detected using an assay without prior separation of the amplicons, they are detected using different detectable molecules to allow the amplicons from the different primer pairs to be distinguishable. For example, probes used to hybridize to the various amplicons can be labeled with labels that are detectable at different wavelengths.

In accordance with another embodiment of the present invention, the amplicon production is monitored in real-time using procedures known in the art (e.g. see U.S. Pat. No. 6,569,627) and using the detection apparatus of the present invention.

It has now been found that rapid thermal cycling within a temperature spectrum can successfully achieve a PCR, and cycling between exact temperatures is not required, In the past PCR has been understood to be a combination of three sequential reactions (i.e. denaturation, annealing, extension) occurring at three different temperatures for three time periods. The present invention makes use of the fact that each of the reactions within PCR can occur over a range of temperatures and these temperatures overlap. Denaturation and annealing each occur so rapidly that no holding time at a particular temperature is necessary for these reactions to occur. Extension occurs over a range of temperatures at varying rates and can occur between the annealing temperature and the denaturation temperature. As a result, the method of the present invention makes use of a single temperature for both the annealing and extension portions of PCR.

Some advantages of the techniques contained herein are based on rapid cycling, with its advantages in speed and specificity.

Technical Description of the Rapid Thermal Alternator

The specification describes the apparatus positioned in a convenient horizontal orientation as illustrated herein. Accordingly, directional references such as “horizontal” refer to the apparatus when in this orientation. The device may be operated in other orientations, with suitable modifications. For convenience of description, terms such as “horizontal” are used but understood as being capable of suitable modification.

The present invention provides a thermal cycler device for performing reactions, such as PCR, using at least two spaced apart temperature blocks, each maintained at a different temperature. Each temperature block is configured to receive one or more reaction vessels such that only a portion of the outer surface of the one or more reaction vessels is in direct contact with the temperature block. As a result of this configuration, the reaction vessel or vessels can be moved from one temperature block to the other using a one-dimensional (e.g. lateral) movement of either the reaction vessel(s) or the temperature blocks, or a combination thereof.

In the past, thermal cyclers having more than one temperature block have required that the reaction vessels be lifted from a receptacle in one temperature block, moved over and lowered into a receptacle in the second temperature block. In the past this design has been used for performing PCR because it was previously believed that the most effective way to achieve precise temperature control of the reaction mixture was to maximize the surface area of the reaction vessel in direct contact with the temperature source. It has now been found that this is not required for efficient PCR. In fact, only a portion of the outer surface of the reaction vessel surface needs to be in direct contact with the temperature block. It is only necessary that a limited amount of outer surface be in contact with the temperature block.

The amount of surface area contact between the heating block and the reaction vessel will influence the rate at which a sufficient portion of the reaction mixture is heated or cooled sufficiently to drive the reaction. The higher the amount of surface area contact, the faster the speed at which the reaction is driven, and vice versa. Faster thermal transfer can also be influenced by other factors, including, but not limited to, the thickness of the reaction vessel wall, the thermal conductivity of the material of the reaction vessel, the thermal conductivity of the heating block, the shape and size of the reaction vessel, the shape of the reaction vessel receiving groove within each temperature block and the volume of the reaction mixture.

The above factors can be altered depending on the intended application of the device of the present invention. Similarly, the amount of direct surface area contact between the outer surface of the reaction vessel and the temperature contact can be varied depending on the intended application. For example, if faster thermal transfer is required then a larger direct surface area contact is required than if a slow thermal transfer is satisfactory.

A standard PCR tube may be used as the reaction vessel and in one version at least about 40% of the outer surface of the reaction vessel (wherein the outer surface does not include the surface of any lid present on the reaction vessel) must be in direct contact with the temperature block. As seen in the figures, an effective region of contact is about 50% of the vessel wall.

In one embodiment, the system contains two temperature blocks mounted in side-by-side spaced apart relation. Transfer of the reaction vessel or vessels between the two reaction blocks is achieved either by one-dimensional (lateral) movement of the vessel or vessels or by one-dimensional movement of the temperature blocks. The reaction vessel or vessels may be held by holding means attached to a horizontal transfer means, such as a robotic arm, that transfers the vessels or vessels via a substantially horizontal movement between the two temperature blocks.

An alternative embodiment composes three or more temperature blocks. As the two-block system, the reaction vessel(s) is moved, or the blocks are moved, such that the reaction vessel is in direct contact with only one temperature block at a time.

As shown in FIG. 1, each heating block 10,20 has a vessel-shaped slot 22 cut into the upper side facing each other that acts as receiving means for the reaction vessel. Each slot 22 may be coated with a low-friction, high heat transfer film such as Teflon™, to ensure uniform and rapid heating of the vessel as it is moved into the slot. The slot 22 is shaped as to completely contact the reaction vessel on all sides except the facing side opposite the other heating block.

The reaction vessel can be a standard conical PCR reaction tube 23 (e.g. 200 μl volume) or alternatively can be a rectangular cuvette. The reaction vessel is preferably designed with a thin-walled material that facilitates heat transfer between the heating block and the reaction mixture within the reaction vessel when a portion of the outer surface of the vessel is in direct contact with the heating block. Preferably the vessel has a rim about the periphery of its top, which provides a seating surface for contact with the holding means attached to the mechanical arm 24. A flat cap that sits partially inside the top of the vessel seals the top of the tube, providing an air tight seal to prevent evaporation. The walls of the vessel are formed to be vertically rigid up to a temperature of about 110° C. to ensure a tight seal with the vessel slot of the heating block 10,20 during heating. The vessel is formed from a transparent material, preferably a plastic such as a polypropylene derivative or glass. Preferably, the vessel has high transmittance of visible light, low vessel wall gas permeability and sterile inner surface.

As shown in FIG. 2, the arm mechanism 24 is designed as to transport the reaction vessel horizontally between the vessel slots of the heating blocks. The arm 24 includes a platform 25 having openings 27 to hold the reaction tubes 23. To move the arm mechanism, a IX” motor 30 with a threaded shaft 32 is attached to a gear or set of gears 34. A lubricating substance may be placed between the gears to increase smooth gear movement. The gearing of the DC motor reduces the effective rotations per minute (RPM) of the motor and provides frictional force to stop and start the arm mechanism. As shown in FIGS. 2 A and B, the arm 24 which supports the reaction vessel is mounted parallel to two circular metal rods 36. On each end of the platform is a semi-circular “U” shaped clip 38 which slideably engages a corresponding rod 36 to the arm 24 to move freely, with minimal friction, along the two parallel circular metal rods 36. Alternatively, as seen in FIG. 1, the clip 38 may fully encircle the rod 36. A lubricating substance can be placed between the “U” shaped attachments and metal rods to reduce frictional force. On one end of the arm 24, a straight gear 40 with teeth is attached to the DC motor gear set 34. As the motor 30 rotates and drives the gear set 34, the shaft 32, by method of frictional movement between gears 34,40, drives the arm 24 along the two parallel metal rods 36. The motor 30 is connected via wires 42 to a circuit and controller which will be described farther below.

As shown in FIG. 2, the purpose of the platform 25 is to hold in place the reaction vessel 23. Located near the middle of the platform 25 is an opening through which the reaction vessel 23 is placed. The rim 44 of the vessel sits directly around the opening to hold the vessel in place. Optionally, as seen in FIG. 3, a flat metal lid 46 is attached and covers the area of the platform which houses the top of the vessel. The metal lid 46 is attached to a hinge 48 and has a clamping mechanism (not shown) to provide pressure on the top of vessel. The pressure of the metal lid on the top of the vessel ensures the vessel is held securely in place and will remain perpendicular to the horizontal orientation of the platform. The metal lid may also have a resistive heating element 50 mounted to its surface. The purpose of the resistive heating element is to transfer heat from the metal lid to the top of the vessel. If the heated metal lid is set to a temperature above the maximum temperature of the vessel, it will minimize condensation of the reaction solvent at the top of the cap of the vessel, which would otherwise increase reactant concentration and potentially adversely affect the formation of PCR products.

The heated metal lid 46 is connected via wires 52 to a circuit and controller which will be described further below. Alternatively, if a heated metal lid is not included, the reaction can be overlaid with a mineral oil, or a similar substance, which would also minimize or eliminate condensation of the reaction solvent on the lid of the vessel. A substance, such as mineral oil, with a boiling point much greater than the reaction solvent reduces evaporation of the reaction solvent.

As shown in FIG. 2, the entirety of the arm mechanism 24 is mounted so that it does not come in contact with the heating blocks 10,20 nor impede the movement of the vessel 23. One such method is by securing the arm mechanism with posts attached to the base and arm mechanism.

A circuit board 53 electrically attached by means of wires 54 to the resistive heating elements attached to each block 10,20, the temperature sensors attached to each block, the motor 30 driving the arm movement, and the microcontroller 26. The circuit board consists of:

-   -   Circuit 1—a current or voltage regulator circuit for the         resistive heating elements     -   Circuit 2—a temperature sensor circuit for the block temperature         sensors     -   Circuit 3—a motor driver circuit for the arm movement motor

The current or voltage regulator circuit, Circuit 1, regulates the current and voltage passed to the resistive heating elements. Circuit 1 can be any well-known current and voltage regulator, such as a MOSFET circuit driver or relay driver. The current and voltage passed to the resistive heating elements is regulated by the microcontroller 26.

The temperature sensor circuit, Circuit 2, can be any well known circuit that responds to a change in voltage, current or resistance transmitted by the temperature sensor. In this embodiment, the temperature sensor circuit consists of a linear resistance input varying with temperature from the temperature sensor, National Semiconductor™ model number LM335AZ, connected to an analog to digital integrated circuit, National Semiconductor model number ADC0831CCN. The digital number representation of the temperature sensor is transmitted via wire to the microcontroller.

The motor driver circuit, Circuit 3, can be any well known motor driver circuit, such as Texas Instruments™ motor driver integrated circuit model number L293D. The motor driver transmits current to the arm motor and is capable of forward and reverse current polarity to move the arm mechanism horizontally back and forth. The motor driver is connected via wire to the microcontroller 26.

The microcontroller 26, controls the overall operation of all circuit components and mechanical parts. In the embodiment of the invention shown in FIG. 2A and FIG. 2B, the microcontroller 26 used is a Parallax BS2-IC™ with the Microchip PIC16C57c™ and a Parallax™ protoboard. The microcontroller 26 receives a digital signal from the temperature sensor circuit which represents the temperature of each block 10,20. The microcontroller is programmed with a predetermined hold temperature of each block. Until the temperature of each block reaches its hold temperature, the microcontroller maintains the current flow through Circuit 1 which drives each resistive heating element. Once each individual heating block 10,20 reaches its respective hold temperature, the microcontroller stops current flow through Circuit 1. As the temperature of each heating block drops below the hold temperature, current flow through Circuit 1 is reactivated. The microcontroller effectively regulates and monitors the temperature of each heating block and maintains temperature uniformity of each heating block.

The microcontroller 26 can be programmed to actuate the motor driver circuit, Circuit 2, at predetermined time intervals, directions and durations. This has the effect of activating the DC motor 30, thereby driving the arm gearing system 34,40. This translates into horizontal movement of the arm platform which shuttles the vessel between the slots of the heating blocks 10,20.

An example operation of the thermal cycling device consists of programming the microcontroller 26 via keypad (not shown) and display (not shown) to hold an individual temperature of each heating block 10,20. The microcontroller 26 is also programmed with a set number of cycles of arm movement. The microcontroller is also programmed with a set dwell time of the vessel as it is moved into the slot of each heating block and as it is located between the two heating blocks.

A sample programmed run of the microcontroller 26 could consist of (A) waiting for heating blocks to reach set temperatures, (B) movement of the vessel by the mechanical arm to the slot opening of the first block, (C) holding the cuvette at the first block for a set period of time (D) movement of the vessel by the mechanical arm to the slot opening of the second block, (E) holdings the vessel at the second block for a set period of time, (F) repeating steps B to E for a set number of cycles, (G) moving the vessel to a location between the two heating blocks for removal.

The thermal cycling device component of the present invention, due to the constant temperature heating blocks, is capable of cycling reaction samples in a vessel through significantly shortened temperature versus time profiles compared to prior art. The device depicted FIGS. 1 and 2 can be used for a two-step DNA amplification reaction. The length of each reaction cycle is significantly reduced in comparison to that observed in standard, single-block thermal cyclers, since there is no temperature ramp-up and ramp-down of the temperature blocks required. The same reaction cycle using prior art devices would take approximately 5-10 times longer because of the ramping times. Decreased cycle times can lead to better yield and specificity of the polymerase chain reaction over prior art cycling. Specifically, in the past it has been found that ultra fast ramping times resulted in improved specificity and increased yield; in PCR amplifications. Rapid cycling results in less time for primer extension at nonspecific annealing sites, consequently, the amount of non-specific product is directly related to the tune at the low temperature, which is the annealing temperature in standard PCR (Wittwer C. T. et al, Biotechniques, 10:76-83 (1991). A rapid cool-down (>5° C./sec) of the PCR mixture favors the kinetics of primer annealing over the thermodynamic advantage of product reannealing. This, in turn, results in an increased product yield.

Furthermore, a shortened time (for example, less than 5 seconds) required to bring the temperature of the reaction mixture from one temperature level to the next temperature level corresponding to phases in the amplification process, is facilitated in the system of the present invention. Specifically, the time is shortened in comparison to ramp times in standard PCR, especially standard PCR performed using a single-block device. The decrease in time required to change the temperature of the reaction, decreases the overall time required for to complete nucleic acid amplification.

The simplicity of the horizontal movement of the mechanical arm system between the two heating blocks, significantly decreases the complexity of control and cost of the thermal cycling device compared to those currently in use. Previous device require complicated robotic arm construction and precise microprocessor control to achieve a similar movement of a reaction sample between heating blocks.

Amplification products obtained through the use of the thermal cycling device of the present invention are qualitatively and quantitatively similar to those obtained through the standard Peltier heating block cycling method. However, advantages in specificity and yield are possible with rapid thermal control of the reaction mixture using the device of the present invention. Such a rapid response is not possible with prior art systems.

By reducing the ramping time of the reaction sample, the present invention can markedly decrease the total time required for the polymerase chain reaction. In addition, the vessel can be designed to hold small reaction samples which reduces the amounts of expensive reagents which must be used thus further reducing the cost of carrying out procedures using the present invention.

The thermal cycling apparatus component of the present invention is useful for amplifying DNA from any source. Although particular configurations and arrangements of the present invention have been discussed in connection with the specific embodiments of the thermal cycling device as constructed in accordance with the teachings of the present invention, other arrangements and configurations may be utilized. For example, various cuvette or heating block configurations may alternatively be used in the thermal cycling device.

As will be appreciated by a worker skilled in the art the thermal cycling device of the present invention provides even greater improvement over the prior art in the speed at which thermal cycling can be carried out, e.g., 30 cycles of DNA amplification in 10-30, or fewer, minutes.

It will be appreciated that the apparatus described herein can readily be used for many different applications including; polymerase chain reaction processes; cycle sequencing; and, other amplification protocols such as the ligase chain reaction, The present invention also advantageously provides an apparatus for accurately controlling the temperature of samples located in the reaction vessel and quickly and accurately varies the temperature of samples located in a vessel according to a predetermined temperature versus time profile.

The configuration of the thermal alternator device of the present invention allows it to be readily combined with detection systems, such as the fluorescence detection system described in more detail below. It should be readily appreciated, however, that the device is not limited to combination with a fluorescence detection system. For example, it can be easily adapted for use with systems, including but not limited to, a visible light detection system, a luminescence detection system or a magnetic detection or separation system. The configuration of the present device permits such adaptation to be well within the abilities of the skilled worker.

Technical Description of the Fluorescence Detection System

As shown in FIG. 3, the device of the present invention optionally includes a fluorescence detection system, which can be located directly between the two temperature heating blocks 10,20 and beneath the horizontal translation means. The fluorescence detection system consists primarily of an excitation source 60 and a detector 62. The excitation source is a light source, which will be described further below, which generates emissions from the sample inside the cuvette 23. The sample fluoresces when illuminated by the excitation source 60. The detector 62 consists of a photosensitive sensor, capable of quantifying the intensity of light produced by the emission source.

The excitation source 60 is located on one side of the vessel 23 at an optimal distance to focus light into the chamber, thereby illuminating the sample. Preferably, the excitation source has a peak wavelength compatible with fluorescent dyes; for example 480 nm. In this embodiment, the excitation source is a blue Light Emitting Diode (LED) with a 3000 mcd at 30 mA and 15-degree focusing angle. The excitation source 60 is enclosed within an opaque tube to prevent excess leakage of light from the source. The excitation light source wavelength is restricted with an optical low pass filter 64 placed directly in the path of light. An optical filter is needed to differentiate the emission from excitation wavelength. In this embodiment, a 500 nm low pass blue dichronic filter is placed in the path of light from the excitation source.

The emission source consists of a fluorescent entity and a nucleic acid amplification product. When illuminated by the excitation source, the entity (for example, a double-stranded DNA specific dye or a fluorescently labeled probe) and nucleic acid amplification product emits light at a different peak wavelength than the excitation source. Examples of suitable fluorescent dyes include, but are not limited to, thiazole orange, SYBR™ GREEN I, ethidium bromide, pico green, acridine orange, YO-PRO-1, and chromomycin A3. Alternatively the fluorescent entity is a nucleic acid probe that is specific for the amplification product and that is labeled with a fluorescent tag.

The detector 62 is located on one side of the vessel 23, directly opposite the excitation source 60, at an optimal distance to collect light from the emission source. Preferably, the detector is a photosensitive sensor capable of differentiation of visible light at a chosen peak wavelength. In this embodiment, the detector consists of a CDS photodiode. The detector is enclosed within an opaque tube to prevent excess light from being detected by the sensor. The wavelength of light detected by the sensor is restricted with an optical filter placed direction in the path of the emission source. An optical filter is needed to differentiate the emission source from the excitation wavelength, hi this embodiment, a 520 nm band pass green dichronic filter is placed in the path of light from the emission source.

Both the excitation source and the detector are activated by means of the microcontroller 26. In this embodiment, when a fluorescence reading of the sample within the vessel is desired, the microcontroller 26 activates the excitation source 60. The excitation source then illuminates the emission source. The light generated by the emission source strikes the detector. In this embodiment, a relative amount of light from the emission translates into a change in resistance of the photodiode detector. This resistance is monitored via wire by the microcontroller. Any change in fluorescence translates into a change detected by the microcontroller.

In a sample run of the fluorescence detection system, a sample within a reaction vessel is placed into the thermal cycling device. A fluorescence measurement is taken at the ambient temperature of the device. Following this measurement, the PCR reaction takes place over a predetermined number of cycles. Following the completion of the PCR reaction, the vessel is positioned between the fluorescence detection system and another measurement is taken. By comparing the initial and final fluorescence of the sample in the vessel, a corresponding increase in nucleic acid amplification product can be determined. Fluorescence measurements may also be taken at the completion of each PCR cycle, thereby quantifying per cycle the relative amount of nucleic acid amplification product increase.

The fluorescence detection component of the present invention, due to the simplicity of design, is capable of measuring per cycle results of nucleic acid amplification product by means of fluorescence. Compared to prior art fluorescence detection systems implemented in heating block thermal cyclers, this device component offers significant reduction in mechanical complexity and cost, while maintaining similar performance capabilities. DNA amplification can be measured by means of fluorescence at the beginning and end of the PCR reaction, as well as during each step. This same performance measurement using prior art system would be approximately 100 times more expensive. The simplistic optics, excitation source and detector of the present invention, have proven also to produce comparable results to more expensive and complex prior art systems.

Furthermore, the rapid cycling of the thermal cycling component means a quantitative fluorescence measurement of nucleic acid product can be accomplished much faster than prior art systems. This greatly reduces the time required to quantify any nucleic acid product generated by the PCR reaction.

Turning now to FIG. 4 and FIG. 5, a thermal cycling device with two fixed temperature heat blocks was constructed based on the principles described in U.S. Patent Application 60/563,061 (which is incorporated herein by reference), but modified as follows as shown in FIG. 4 and FIG. 5.

A holder 100 for the reaction vessels 120 was constructed by drilling holes of the appropriate size into a chassis 140 comprising a flat sheet of metal. The reaction vessel holder 100 was bolted onto chassis 140. Underneath the reaction vessel holder 100 proximal heater block 160 a, and distal heater block 160 b were affixed to a support board 180. Grooves (not seen) were machined into the proximal heater block 160 a, and distal heater block 160 b, the grooves being shaped precisely to fit the shape of the reaction vessels 120. The proximal heater block 160 a, and distal heater block 160 b also contained resistive heaters (not seen) which were controllable to maintain a set temperature. The support board 180 was configured and arranged to be able to move in one dimension by sliding along two metal shafts 120. Motion of the support board 180 was driven by a cam shaft 122 which was configured and structured to be rotatable in one direction by a motor (not seen). The cam shaft 122 was thus configured to rotate in between a pair of plastic or metal leaf springs 124. The cam shaft 122 was configured and structured to have three main positions, namely: (1) pointing parallel away from the proximal heater block 160 a, and distal heater block 160 b to result in a configuration where the distal heater block 160 b came into contact with the reaction vessels 120; (2) pointing perpendicular to the proximal heater block 160 a, and distal heater block 160 b to result in a configuration where neither of the proximal heater block 160 a, and distal heater block 160 b were in contact with the reaction vessels 120; and (3) pointing parallel towards the proximal heater block 160 a, and distal heater block 160 b to result in a configuration where the proximal heater block 160 a came into contact with the reaction vessels 120.

Thus, when the cam shaft 122 were in the 2^(nd) position, the reaction vessels 120 are not in contact with either the proximal heater block 160 a, nor the distal heater block 160 b. If the middle section 126 were to be cut out of the support board 180, then this middle position 126 would be convenient for imaging the bottom of the reaction vessels 120. Specifically, a blue LED light source may be shone at the bottom of the reaction vessel to excite the contents of the vessel, e.g. SYBR® Green Dye (Molecular Probes, Inc.). Emitted light from the vessel may be detected by means of a CCD camera. To filter out blue light from the LED source, a bandpass filter may be placed in front of the CCD camera so that only higher wavelengths e.g., green and red are allowed to pass through. This helps improve the signal-to-noise ratio.

The use of a cam shaft 122 with the leaf springs 124 attached to the support board 180 helps ensure good contact between the proximal heater block 160 a, and distal heater block 160 b and the reaction vessels 120. The reason is because the cam shaft 122 is able to deflect the leaf springs 124 when the cam shaft 122 is parallel to, and facing either towards or away from the proximal heater block 160 a, and distal heater block 160 b. This enables the cam shaft 122 to exert extra force, thereby to drive the proximal heater block 160 a, and distal heater block 160 b into contact with the reaction vessels 120, and to correct for dimensional tolerances.

It is important to note that the according to certain aspects of the present invention the reaction vessels 120 only come into partial contact with the proximal heater block 160 a, and distal heater block 160 b. This means that there is a non-uniform (i.e. nonzero) temperature gradient across the reaction vessel 120. The reason is because, although the proximal heater block 160 a, and distal heater block 160 b are set at a certain temperature, the top of the reaction vessels 120 experience a different temperature because it is held in place by a material which serves as a passive insulator, and the side walls of the reaction vessels 120 which are not in contact with the proximal heater block 160 a, and distal heater block 160 b are exposed to the temperature of the ambient air.

FIG. 6 shows the control and processing electronic block diagram of the apparatus of FIGS. 4 and 5 (depicted pictorially in FIG. 6)

The steps through which the microprocessor (μP) shown in FIG. 6 goes are shown in FIG. 7. These steps are accomplished as shown in more detail in FIGS. 8A and 8B. Once the samples have been prepared and loaded into the holder 100 (FIG. 4) and the operator gave the “start” signal (e.g. by pressing a button or key), the μP is in control of the entire session as shown in FIGS. 8A and 8B. At the end, each of the four reaction vessels or “cuvettes” 12 is tested for sufficient fluorescence, which indicates presence of the target DNA, and the result is acquired and stored/displayed accordingly, and test completion is indicated.

It will be understood by those skilled in the art that the embodiments described herein are merely exemplary and that the person skilled in the art may make many modifications and variations without departing from the scope of the invention. The various embodiments may be practiced in the alternative or in combination as appropriate. All such variations and modifications are intended to be included within the scope of the invention. 

1. In a thermal cycling process for polymerase chain reaction (PCR) wherein a reaction vessel (RV) having a vessel wall—containing a reaction mixture comprising a target nucleic acid (NA) and reagents selected to achieve amplification of said target NA by means of the PCR—is thermally cycled between at least two predetermined temperatures, the improvement characterized by maintaining a temperature gradient within said RV during said thermal cycling process.
 2. The process defined in claim 1, wherein said temperature gradient is obtained by applying heat unequally to the wall of said RV.
 3. The process defined in claim 2 wherein said heat is applied unequally to said vessel wall by contacting only a portion of said wall with a heating element while exposing the remainder of said wall to ambient air.
 4. The process as defined in claim 1, further characterized by rapid thermal cycling wherein a temperature spectrum of said NA and reagents is maintained within said RV during the thermal cycling process.
 5. The process as defined in claim 1, further characterized by annealing and extension of said NA occurring in a single step during the PCR process.
 6. The process as defined in claim 1, further characterized by RV temperature ramp-time in excess of 2° C./sec.
 7. The process as defined in claim 1, further characterized by real time fluorescence-based measurement of said NA amplification, if present in said reaction mixture, during the PCR process.
 8. The process as defined in claim 1 wherein said temperature gradient consists of a large gradient.
 9. A polymerase chain reaction (PCR) device for thermal cycling of a reaction mixture contained in a reaction vessel (RV) having an outer wall, comprising at least two heating sources, means for maintaining said heating sources at different predetermined temperatures, and means for causing said RV to be in successive physical contact with each said heating sources for predetermined times and for a number of successive contacts sufficient to promote the PCR in said reaction mixture contained in said RV, characterized by said heating sources being configured for successively contacting only part of said outer wall of the RV while the remainder of said surface is exposed to ambient air.
 10. The PCR device as defined in claim 8, wherein said at least two heating sources comprise spaced apart heating blocks each having a groove contoured to receive and provide thermal contact with only a portion of the outer wall of the RV that contains the reaction mixture.
 11. The PCR device as defined in claim 8, further comprising means for real-time florescence-based measurement of a preselected nucleic acid (NA), if present in said reaction mixture, during operation of the PCR device.
 12. The PCR device defined in claim 8 further comprising a controlling means for operating said device so as to achieve a time versus temperature profile which maintains a large temperature gradient within said RV during said thermal cycling process.
 12. The PCR device defined in claim 11, wherein said controlling means controls the device to provide annealing and extension of said NA within said RV in a single step during the PCR process by maintaining essentially no holding time at the temperature required for said annealing and extension step.
 13. The PCR device defined in claim 9, wherein said at least two heating blocks are in spaced apart opposing relation to each other and said grooves oppose each other.
 14. The PCR device defined in claim 13, further comprising a holder for retaining said RV such that when engaged within said groove, and drive means for sequentially moving said RV between said blocks for successive contact with said opposing grooves.
 15. The device defined in claim 9, wherein said grooves are dimensioned to contact between about 40% and 50% of the wall of said RV. 